Mammalian hydroxysteroid dehydrogenases (HSDs) play pivotal roles in the biosynthesis and inactivation of steroid hormones. Molecular cloning indicates that HSDs belong to at least two distinct protein families the short-chain alcohol dehydrogenases, and the aldo-keto reductase (AKR) super-family. Rat liver 3alpha-HSD(EC 1.1.1.50) is a member of the AKR family and inactivates circulating androgens, progestins and glucocorticoids. The cDNA for 3alpha-HSD has been cloned, sequenced and over-expressed in E. coli. The crystal structure of the apoenzyme has been solved at near atomic resolution (3.0 A) and represents the first 3- dimensional structure of a mammalian HSD. The 3alpha-HSD structure adopts a (beta/alpha)8 barrel motif (triosephosphate isomerase barrel) and lacks a Rossmann fold for binding NAD(P)(H). To relate structure to function the following studies are planned. X-ray crystallographic structures of binary [E.NADPH] and ternary complexes [E.NADPH.Steroid] of 3alpha-HSD are now sought at atomic resolution. The existing 3.0 A crystal structure implicates: a catalytic triad in oxidoreduction (Tyr55, Lys84, and Asp50); residues in binding pyridine nucleotide in an extended anti-conformation (Tyr216, Asn167, Ser166, Arg270 and Arg276); and an elliptical hydrophobic pocket for binding steroid hormone (Tyr55, Trp86, Phe118 Phe128, Phe129 and Trp227). These residues will be mutated. Site-specific effects will be distinguished from global effects by comparing the kinetic and spectral fingerprints of r3alpha-HSD with mutant proteins. Changes in kinetic parameters (Km, kcat, kcat/Km and pH-kcat profiles), and ligand affinity (cofactor or steroid) will determine which amino acids are important in catalysis and which are important in ligand binding. To verify atomic models for binding NADPH to 3alpha-HSD this site will be affinity-labeled with adenosine [e.g., 3'- and 5'-p- (fluorosulfonyl)-benzoyl-adenosine] and nicotinamide [e.g., haloacetyl- pyridine] derivatives and modified amino acids identified. Secosteroid mechanism-base inactivators will be used to label amino acids involved in steroid recognition. These secosteroids lack A and D rings but contain acetylenic alcohols at the equivalent of either the C-3 [1,10-seco-5alpha- estr-1-yne-3(R,S)-17beta-diol] or C-17 [17(r,S)-hydroxy-14,15-secoestr-4- en-16-yn-3-one] positions. Both these alcohols are oxidized by 3alpha-HSD to the corresponding acetylenic ketones which covalently modify the enzyme. Since oxidation is catalyzed at both the C-3 and C-17 positions one of these partial steroids must enter the active site backwards. To identify amino acids tagged by the acetylenic ketones, radiolabeled inactivated enzyme will be subjected to peptide mapping and sequencing. Our studies will provide a structural basis by which HSDs recognize their ligands and inactivate steroid hormones and may be applicable to all HSDs that belong to the AKR super-family.